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Biomedical Research

Effect of Oxidative stress on sperm quality in Leukocytospermic infertile men

Author(s): Garg Vidya, Garg S.P.,Rawekar A.T., Deshpande V.K., Biswas D.A., Sawane M.V., Akarte A.N.

Vol. 22, No. 3 (2011-07 - 2011-09)

Garg Vidya1, Garg S.P.2, Rawekar A.T.3, Deshpande V.K.3, Biswas D.A.3, Sawane M.V.2, Akarte A.N.4

(1) Department of Physiology, S. S. Medical College, Rewa (M.P.), India
(2) Department of Forensic Medicine, Bundelkhand Medical College, Sagar (M.P.), India
(3) Department of Physiology, J.N. Medical College, Sawangi (Meghe) Wardha (M.S.), India
(4) Biochemistry), Department of Biochemistry, J.N. Medical College, Sawangi (Meghe), Wardha (M.S.), India


It has been proposed that leukocytospermia has a role to play in male infertility. Leukocytes are considered to be the primary source for generation of reactive oxygen species (ROS) re-sulting in oxidative stress. The aim of this study was to evaluate the effect of oxidative stress created by leukocytospermia on sperm parameters and sperm function test. Specimens were divided into two groups: Group 1. Non-leukocytospermic WBC ≤106 /ml (N=60) Group 2. Leukocytospermic WBC>106 /ml (N=56). Leukocyte measurement was done by Peroxidase staining using Ortho-Toluidine (WHO, 2000). Semen parameters in group 1 and 2 infertility subjects were studied. Sperm concentration, sperm motility (%), normal sperm morphology (%), % Viability, HOS (%), MDA and NO concentration were compared in two groups i.e. non-leukocytospermic and leukocytospermic subjects. MDA and NO concentration were higher in leukocytospermic than nonleukocytospermic subjects and they had statistically significant difference (p<0.0001). Data from current study suggest a possible role of oxida-tive stress on sperm quality in leukocytospermic infertile men.

Key words: Male infertility, Leukocytospermia, Oxidative stress
Accepted February 27 2011


Many environmental, physiological and genetic factors have been implicated in poor sperm function and infertil-ity. Although there are some definite causes for male in-fertility still the term “Idiopathic Infertility” remains. Various studies are going on effect of oxidative stress on fertility potential of male which can be one of the causes of idiopathic infertility.

In recent years, the generation of reactive oxygen species (ROS) in male reproductive tract has become a real con-cern because of their potential toxic effects at high levels on sperm quality and function. Reactive oxygen species (ROS) are highly reactive oxidizing agent belonging to class of free radicals [1].

When levels of reactive oxygen species overwhelm the body’s antioxidant system, oxidative stress (OS) occurs. Spermatozoa are sensitive to oxidative stress (OS) be-cause they lack cytoplasmic defenses [2].

There are two main sources of ROS in semen, leukocytes and immature spermatozoa [3]. Of these, leukocytes are considered to be the primary source. Reactive oxygen species (Malondialdehyde MDA) and reactive nitrogen intermediates (NO Nitric Oxide) are produced by acti-vated macrophages and granulocytes, and adversely affect sperm motility and fertilization functions [4]. Much con-troversy surrounds the clinical significance of an in-creased concentration of white blood cells (WBC) in male ejaculate. The world health organization classification of leucocytospermia is a concentration > 1×106 WBC/ml[5].

Study was conducted at: J.N. Medical College, Sawangi (Meghe), Wardha (MS), India

ROS causes infertility by two principal mechanisms. First, ROS damages thesperm membrane which in turn reduces the sperm’s motility andability to fuse with the oocyte. Secondly, ROS directly damagesperm DNA, compromising the paternal genomic contribution tothe embryo [6].

Considering the all above detrimental effects on various seminal parameters, present study has been carried out to explore the possible effect of oxidative stress on sperm quality in leukocytospermic infertile men.

Material and Methods

Semen samples

This study has been carried out in the reproductive biol-ogy unit (infertility clinic) in department of physiology, Jawaharlal Nehru Medical College Sawangi Wardha (M.S.) after taking approval from ethical committee. The patients were referred to semen analysis laboratory from department of obstetrics and gynecology, department of surgery and sex and marriage counseling centre, J.N.MedicalCollege, Sawangi (M), Wardha.

The subjects included were 116 infertile men in the age group of 23 to 43 years. With due consent of the subjects, their semen analysis was performed. They were all non smokers, non alcoholics and free of any obvious genital tract abnormalities. Instruction regarding abstinence was given for sexual intercourse as well as for masturbation and night emission for period of 3 to 5 days for full matu-ration of sperms. Samples were obtained by masturbation or coitus interruptus method. Patients were advised not to use ordinary condoms, since they may contain spermi-cidal agents.

The samples were allowed to liquefy at least for 30 min-utes protecting it from extremes of temperature i.e. below 20 °C and above 40 °C. The routine semen analysis was carried out after liquefaction with in one hour as per the guidelines laid down byW.H.O, 2000[7, 8]. Each sample was subjected to sperm function test and leukocyte count immediately except biochemical assays.

The study involved thorough (i) history taking, (ii) clini-cal examination followed by (iii) detailed semen analysis (iv) sperm function test (hypo-osmotic swelling test) and finally (v) the MDA and NO level measurement. In de-tailed semen analysis physical and microscopic analysis was done. Physical characteristics of seminal analysis included volume, viscosity etc. Microscopic examination comprised of sperm count in million per milliliter, motile sperm count in million per milliliter, percentage motility, percentage normal morphology and percent viability; known as routine or conventional parameters of semen analysis.

Sperm function test was carried out by hypoosmotic swelling test [9]. Healthy viable sperms which were hav-ing good fertilizing potential had the property of curling of tails when exposed to hypoosmotic solution. The un-healthy poor quality sperm do not have curling. If more than 60% spermatozoa, shows curling then it is consid-ered as normal.

Leukocyte measurement was done by Peroxidase staining using Ortho-Toluidine as per the guidelines laid down by W.H.O, 2000 [7, 8]. After centrifugation seminal plasma was used for meas-urement of free radical production by estimating malondialdehyde [10] and assay of nitric oxide by Griess reaction of kinetic method in which nitrate is reduced to nitrite by copper coated cadmium granules [11]. Concen-tration of MDA and NO were measured spectrophotomet-rically by using UV-VIS spectrophotometer (systronics, model no.-117). Results of oxidants and leukocytospermia were compared with different seminal parameters. Statistical analysis of the results was done. The results were compared with those of the other studies.

Statistical Analysis

The data obtained was analyzed by using mean ± standard deviation and student unpaired ‘t’ test. The results were tested by using the SPSS (Version 14.0) statistical soft-wares.


Specimens were divided into two groups: group 1. Non-Leukocytospermic WBC ≤106 /ml (N=60) group2. Leu-kocytospermic WBC>106 /ml (N=56).

It was observed that MDA and NO concentration were significantly higher in leukocytospermic than nonleuko-cytospermic subjects (p<0.0001) (Table1).

Routine seminal parameters and sperm function test (HOS %) were significantly lower in leukocytospermic than nonleukocytospermic subjects (p<0.0001) (Table1).

Table 1: Routine Seminal parameters, Sperm Function Test (HOS) and Biochemical parameters (MDA & NO) in Non-Leukocytospermic WBC ≤106 /ml (N=60) and Leukocytospermic WBC>106 /ml (N=56) infertile subjects.

Semen parameters White Blood Cell Concentration
WBC ≤106/ml (N=60) WBC>106 /ml (N=56)
Sperm Concentration (million/ml) Mean± SD 61.66±26.99 23.55±23.08
0.000, HS, p<0.0001
% motility Mean± SD 47.20±9.33 39.33±8.81
0.000, HS, p<0.0001
% Morphology Mean± SD 46.93±14.33 24.51±13.63
0.000, HS, p<0.0001
% Viability Mean± SD 70.66±9.88 58.32±13.61
0.000, HS, p<0.0001
% HOS Mean± SD 70.73±9.11 46.30±12.62
0.000, HS, p<0.0001
MDA Mean± SD 5.32±2.53 8.75±2.87
0.000, HS, p<0.0001
Nitric Oxide (μmole/ml) Mean± SD 33.26±12.21 45.87±11.91
0.000, HS, p<0.0001

Data shown above is of 116 subjects excluding Azoospermic and Necrozoospermic subjects. All parameters were com-pared in two groups nonleukocytospermic and leukocytospermic and they had statistically significant difference (p<0.0001).


There are several evidences demonstrating that WBCs may affect seminal quality and fertility [12, 13]. It has been confirmed that men having leukocytes >1×106/ml have lower sperm motility, viability and other parameters.

In the present study, higher levels of oxidants (MDA and NO) and lower levels of sperm parameters as well as sperm function test suggest that leukocytes may be re-sponsible for increase in oxidants (MDA and nitric oxide) which may have deleterious effect over seminal quality. This may be because the leukocytospermic sample had higher ROS production resulting in oxidative damage.

Male genital tract inflammation typically results in leuko-cytospermia, of which approximately 82% leukocytes are granulocytes and small number of lymphocytes (T & B cells) and monocytes / macrophages may also be present. In semen granulocytes are major producer of reactive oxygen species (ROS). The sperm plasma membrane con-tains large amounts of lipids in the form of polyunsatu-rated fatty acids which trigger a chain of chemical reac-tions called lipid per oxidation in presence of ROS [14]. MDA (malondialdehyde) is most widely used tool to as-sess lipid peroxidation.

Release of nitric oxide by lymphocytes has also been re-ported. NO may play a part in tissue damage, for it may be cytostatic or cytotoxic not only for invading microor-ganisms but also for the cells that produce it and for the neighboring cells. In some situations it may interact with oxygen derived free radicals to generate molecules that could enhance its cytotoxicity [15].

In a study, Plante et al [16] found that although the ex-tracellular production of ROS by deficient spermatozoa was inadequate to compromise the motility of normal spermatozoa but more than one million activated poly-morphonuclear neutrophils per ml with a one thousand fold higher ROS production; had detrimental effects on the motility of normal washed spermatozoa.

Baker et al [17] demonstrated, on a large number of sam-ples, the negative correlation between reactive oxygen species generation by leukocytes and sperm motility. Saleh et al [18] hypothesized that seminal leukocytes may play a role in stimulating excessive ROS production by spermatozoa.

Novotny et al [19] studied the occurrence of ROS in se-men of 68 males of infertile couples. They suggested that spermatozoa might be the source of ROS as well as the seminal leukocytes.

Yadav SB et al [15] studied 83 semen specimens and they were grouped as nonleukocytospermic and leukocyto-spermic samples. Leukocytospermic samples had signifi-cantly high levels of nitric oxide along with defective sperm function (HOS) was observed. Sperm concentra-tion, % motility was also lower in leukocytospermic sam-ples.

However, certain contrast studies have shown favorable effect of leukocytes on human spermatozoa [20, 21, 22].

Our results indicate that the diagnostic and prognostic capabilities of the seminal oxidative stress (OS) test are beyond those of conventional tests of sperm quality and function. The OS test can identify patients with a clinical diagnosis of male factor infertility. It is strongly recom-mended that incorporating such a test into the routine an-drology workup is an important step for the future of the male infertility practice.


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Correspondence to:
Vidya Garg

E-14, Doctor’s Colony
Rewa (MP) 486001, India

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